The Gram-positive bacterium Streptococcus pyogenes (group A streptococcus) is one of the most important pathogens that can infect the skin where it causes numerous diseases ranging from the relatively superficial impetigo to life-threatening necrotizing faciitis. Since it is likely that an encounter between an keratinocyte and S. pyogenes represents an early event of all streptococcal skin disease, an analysis of the molecular basis of the interaction and its consequences will provide a powerful model for understanding the contribution of the keratinocyte to defense against microorganisms. Initial studies have revealed that the manner by which keratinocytes sense and respond to the presence of S. pyogenes is influenced through the expression of at least two streptococcal proteins. The first is an adhesin known as M protein that directs attachment of the bacterium to the keratinocyte and the second is streptolysin O (SLO), a membrane-active pore-forming toxin. Furthermore, expression of the genes that encode these streptococcal proteins is highly regulated by S. pyogenes. Preliminary evidence suggests that expression of SLO is developmentally regulated and may involve a bacterial autocrine factor. An understanding of how bacterial and host cell regulatory networks interact to influence the course of infection will provide insight into how keratinocytes help to orchestrate host defense responses. This proposal will examine the regulatory events involving SLO and their effect on infection of keratinocytes by: 1.) Identification of the streptococcal regulatory genes which control expression of SLO. This will be essential for understanding the signals by S. pyogenes in the skin. 2) Analyzing how the regulation of SLO and expression of the adhesin interact to induce distinct patters of keratinocyte responses in existing cell culture models; and 3.) Examining the role of SLO and the adhesin in manipulating keratinocyte function in an advanced organotypic model of streptococcal skin infection. This latter aim well-defined utilize mutant streptococci and the resource of this Program's Histology Core. This project will combine Dr. Caparon's knowledge of streptococcal genetics with the expertise of the other members of this Program in the biology of the keratinocyte. This collaboration is an important component of this program, which will be valuable for understanding the behavior of keratinocytes under any circumstance when they are exposed to bacteria.